Spontaneous damage of DNA bases is a major source of genetic instability. Failure to correct this damage leads to somatic mutations that underlie many diseases associated with aging. The ability to safeguard against these spontaneous lesions relies largely on the base excision repair (BER) pathway whereby DNA glycosylases scan the genome to locate and excise base lesions. Many common forms of damage and proteins involved in the BER pathway have been identified and structurally characterized, but there is a fundamental gap in our understanding of how these enzymes accomplish this amazing task of genome-wide repair. Until this gap is filled, studies of DNA damage and mutagenesis will be largely observational and we will not be able to predict the effects of polymorphisms or exposure to novel carcinogens. Our long-term goal is to understand the molecular mechanisms and fundamental principles by which BER proteins locate and selectively act on a wide range of DNA lesions within genomic DNA. The central hypothesis, that protein-DNA dynamics are critical to the function of the BER pathway, particularly to those enzymes that recognize multiple forms of DNA damage, is strongly supported by data from our lab and others working in this area. Guided by progress and preliminary data from the past funding period, we propose to pursue four specific aims: 1) Determine the searching mechanism(s) of human DNA glycosylases in vitro; 2) Evaluate the hypothesis that facilitated diffusion contributes to efficient DNA repair in eukaryotic cells; 3) Quantify the catalytic specificity of AAG by employing competition kinetics with a wide variety of structurally diverse base lesions; 4) Determine the molecular mechanisms by which AAG achieves efficient recognition of a broad range of substrates. By combining the results from pre-steady state enzyme kinetics, fluorescence spectroscopy, and structure-activity relationships we are poised to dissect the protein-DNA dynamics important for recognition and repair of damaged bases. The approach is innovative, because we are developing novel biochemical assays and new molecular models to understand the kinetic and thermodynamic mechanisms of DNA repair. The proposed research is significant, because it has a high probability of expanding our understanding of BER mechanisms and to uncovering fundamental principles that are relevant for other DNA-templated biological processes. As BER is a critical component of the cellular defense against genomic instability, including endogenous damage, these studies will contribute both to our understanding of mutagenesis and the normal aging process.